Project Summary DNA double stranded breaks (DSBs) are deleterious lesions that require rapid repair to avoid the loss of genetic information, genomic instability, neoplastic transformation and cancer formation. In human systems, the two predominant pathways for double stranded DNA break repair are canonical non-homologous end joining (cNHEJ) and homologous recombination (HR). The cNHEJ machinery recognizes breaks, indiscriminately joins them independent of sequence context and is therefore considered error prone and potentially genotoxic. HR relies on resection of the 5? strand with the generation of single stranded 3? overhangs, which invade homologous sister chromatids to promote error-free break repair. Choice between HR and cNHEJ depends primarily on the cell cycle stage and the nature of the break. During G1 of the cell cycle HR is inhibited by RIF1 and 53BP1, which prevent the required BRCA1/2 complex assembly and end resection for HR initiation. During S and G2, when sister chromatids are available as a template for HR, both cNHEJ and HR pathways can be employed and compete to repair DSBs. End resection is activated by CtIP in S and G2 phases and promotes HR, but it is unclear how the abundant and efficient cNHEJ machinery is suppressed in S and G2 to allow resection at break sites and commencement of HR, thereby ensuring error- free repair of lesions to preserve genome integrity. CYREN (Cell cYcle REgulator of NHEJ) was originally identified in a screen for potential modulators of retroviral infection. Later, an alternatively spliced isoform of CYREN, CYREN-2, was found as short open reading frame translated polypeptide and shown to interact with the Ku70/80 heterodimer, pointing at a potential role in cNHEJ. Here it is proposed to investigate the discovery that CYREN modulates the cell cycle dynamics of cNHEJ and that the small protein is a direct cell cycle regulator of cNHEJ. In three specific aims it is proposed to investigate the mechanism of how CYREN inhibits cNHEJ through the CYREN interaction with the Ku heterodimer complex (AIM 1), how CYREN is cell cycle regulated and controls the cell cycle regulation of DSB repair pathway choice (AIM2) and finally what the effects of CYREN deletion and overexpression are, how the deregulation of cell cycle control of cNHEJ influences genome maintenance and genome instability and whether cells that lack CYREN are sensitive to DNA damage causing agents (AIM 3). In summary, this grant proposal focuses on CYREN, a novel regulator of DNA repair pathway choice, the mechanism of how CYREN is regulated and controls cNHEJ, whether lack of CYREN causes genome instability and whether CYREN targeting can be exploited to sensitize cancer cells to treatment with genotoxic agents.